We are working to establish an in vitro system for the study of checkpoint activation by various reagents that induce DNA damage in human cells. These DNA damaging agents are widely used in chemotherapy. Thus, our research focuses on molecular mechanisms by which these chemotherapeutic agents confer cytotoxicity as well as pathways by which cancer cells achieve tolerance to chemotherapeutic approaches. As a model system, we are investigating checkpoint activation mediated by the DNA mismatch repair proteins MutSalpha and MutLalpha in response to several DNA damaging agents including SN1 alkylators, fluorouracil, and cisplatin. DNA damage sensors that target specific types of DNA damage activate the ATR and ATM protein kinases. This, in turn, activates a cascade of signaling kinases including Chk1 and Chk2 that ultimately result in cell cycle arrest at the G2/M boundary. We are purifying recombinant human proteins involved in this damage response with the goal of defining the underlying molecular mechanism. We are also interested in identifying components of key protein complexes that function to license cell cycle arrest in response to DNA damaging agents. Finally, we are initiating studies to understand how nuclear architecture in the cell can modulate the DNA damage response.